MPL P106L exhibits lower activity than MPL WT. (A) UT-7 cells proliferation in response to THPO: UT-7 MPL WT, UT-7 MPL P106L, UT-7 MPL P106L clone 1 and 5, and UT-7 MPL R102P cells were seeded at the same concentration (1 × 105/mL) after cytokine deprivation and stimulated with various concentrations of THPO compared with the control (GM-CSF). Cell proliferation is expressed in relative percentage compared with the control representing 100%. Viable cells were counted at different time points using KOVA slide. (B) UT-7 cell apoptosis in culture with THPO: cells were cultured for 3 days with various concentrations of THPO, and the percentage of apoptotic cells (Annexin V–positive) was analyzed by flow cytometry using Annexin V assay (105 cells analyzed per condition). (C) UT-7 cells phenotype in response to THPO: cells were cultured with various concentrations of THPO, and CD41a expression was analyzed by flow cytometry after 3 days. At least 3 × 104 cells were analyzed. (D) UT-7 cells signaling in response to THPO: cells were deprived for 5 hours and then stimulated with various concentrations of THPO compared with GM-CSF and analyzed by western blotting at different time points. STAT1, STAT3, STAT5, AKT, and ERK1/2 phosphorylation was examined. Cell-surface MPL expression for UT-7 MPL WT, MPL P106L, MPL P106L clone 1 and 5 correspond to Figure 3A. Intracellular MPL expression corresponds to Figure 3C. *P < .05; **P < .01; ***P < .001.
